Method for recovering and purifying trophoblastic beta-1-glycoprotein

FIELD: medicine.

SUBSTANCE: method for recovering and purifying trophoblastic beta-1-glycoprotein (TBG) from retroplacental blood serum by ammonium sulphate fractionation, washing and centrifugation of the solution residual matter, affinity sepharose chromatography followed by elution and lyophilisation of an end product. The retroplacental blood is centrifugated at 10000 rpm within one hour. Sodium chloride is added to a supernatant to end concentration 0.1-1.0 mol/l, while triton X-100 is added to end concentration 0.01-0.9%. Centrifugation is performed once again. The prepared serum is saturated with ammonium sulphate to 30%, incubated within a night in stirring, centrifugated. The deposit is threefold washed in 30% ammonium sulphate, suspended in 0.15M phosphate buffered saline pH 7.0-7.5, containing 0.5 M sodium chloride and 0.05% triton X-100, dialysed in an ultrafiltration cell, with tenfold amount of the buffered saline passed, five-fold diluted and ultrafiltered at filtration limit 100000 D. The prepared permeate is processed with single affinity sepharose chromatography with immobilised monoclonal TBG antibodies. The affinity-bound TBG is eluted with 0.1M glycine-Hcl buffer solution pH 2.55. The eluate is immediately neutralised, concentrated, dialysed against physiological saline and lyophilised.

EFFECT: higher yield and purity of the end product.

2 ex, 1 tbl

 

The invention relates to pharmaceutical industry and can be used in medicine, biotechnology for producing drug USSR β-1-glycoprotein (TBG), used as a component of diagnostic systems and tools for the treatment of cancer, autoimmune and allergic diseases. The method allows to increase the yield and purity of the target product.

It is known that TBG, as a specific marker of pregnancy, is synthesized by the syncytiotrophoblast cells and secreted into the bloodstream of the mother. According to most authors TBG is a glycoprotein containing up to 28% carbohydrates and having a molecular weight of 90000 Yes (Bohn N., 1974) to 113000 Yes (Tatarinov US and others, 1974). However, in the literature there are data that TBG forms a family consisting of 4 proteins with a molecular mass of 72, 64, 62 and 54 kDa (Plouzek S.A., J.Y. Chou, 1991). Other authors believe that TBG is a heterogeneous protein heterogeneity due to the presence of two options TBG - alpha and beta (Pola A. et al., 1992). When this molecular weight TBG-alpha equal to 110 KD, and TBG-beta 75 KD (M. Ito et al., 1981). The absence of well-attested data about the molecular heterogeneity of TBG and accurate description of the structure of the molecule is largely the reason for the lack of an effective system of selection and ochistitel to obtain TBG is retroplatsentarno blood.

Known multi-step allocation method of TBG retroplatsentarno human blood [1]. Serum obtained from retroplatsentarno blood was fractionally consistently with rivanol and ammonium sulfate, and then subjected to gel filtration on Sephadex G-25. The resulting material was subjected to ion exchange chromatography on DEAE-cellulose. The fractions containing TBG, United, fractionally ammonium sulfate and were gel-filtered on Sephadex G-200. After another alpacamania fractionation and dialysis was performed chromatography on a column of hydroxylapatite and after clarification by centrifugation, ion-exchange chromatography on microgranular cellulose KM-32. The final stage of purification was the preparative isoelectrofocusing on impaling with a range of 3.0 to 10.0. The result of the entire purification procedure was to obtain the drug TBG with purification efficiency of 93%. The yield was 1.1%.

The cleaning procedure allowed to obtain 1.8 mg relatively clean of the desired protein from a sample containing 160 mg, involves two procedures gel-filtration, two ion-exchange, one hydrophobic chromatography, three procedures alpacamania fractionation, isoelectrofocusing.

Thus, the apparent disadvantages of the described method are extraordinary complexity and trudem is here, very high cost, especially considering the extremely low yield of the target product, equal to 1.1%.

There is a method of allocation of TBG waste of gammaglobulin of retroplatsentarno blood [2], providing for the suspension of a solid residue containing TBG in water, centrifugation and subsequent alpacamania fractionation. After 4 days of dialysis and centrifugation of the resulting supernatant is treated with hydroxyapatite and Proskurov 30-60 minutes, and again centrifuged. The supernatant is subjected to concentration ultrafilter and the two-day dialysis, then centrifuged and lyophilizers. Dried product is dissolved in phosphate buffer and subjected to column chromatography on hydroxyapatite. The eluate cialiswhat within 36 hours and lyophilizers. The output is 35.5%, purity of the drug - 95%. The described method is superior to previous and purity of the drug, and exit, but also very time-consuming, takes significant time and leads to unnecessary losses due to the numerous procedures of dialysis.

The same problems burdened methods proposed Hao et al. [4] and Wasiliana et al. [5]that when the purity of the target product (95%) are accompanied by significantly lower yields (20%).

There is a method of extracting the Oia TBG serum retroplatsentarno blood or sediment, obtained in the production of gamma globulin from the serum retroplatsentarno blood [3], providing alpacamania fractionation followed by dialysis for 4 days. After centrifugation the supernatant is subjected to ion-exchange chromatography. The eluate concentrate and cialiswhat within 72 hours. Otvetsvennyy the preparation is centrifuged and passed through the sorbent, containing lectin (concanavalin a). Elyuirovaniya fraction containing TBG, dialist and lyophilizers. The release of the drug in the purity of 96% is 20%.

In the so-called BIC options described ion-exchange chromatography on hexylsilane laid high quantitative loss of protein. The duration of the method, and the low yield of the target product does not allow to consider the described method as a solution to the problem of isolation and purification of this protein complex as USSR beta-1-glycoprotein.

The closest analogue may be considered the method proposed Wageline et al. [5]. The authors used as source material retroplatsentarno blood or residue obtained in the production of gamma globulin. To reduce procedure time, the authors received alpacamania residue, which was subjected sequentially gel-filtration chromatography on Sephadex is -100, ion-exchange chromatography on DEAE-cellulose and chromatography on sepharose with immobilized zibarro-blue dye. At the end of the process, the resulting product was again subjected to gel filtration on Sephadex G-200. The authors rash called chromatography on zibarro-sepharose affine, while is hydrophobic procedure. The multiple steps involved in the extraction procedure determines its complexity and low yield of the target product (about 25%) with good levels of purity (not less than 96%).

An object of the invention is to increase the yield and purity allocated TBG. This object is achieved as follows.

Proven in the absence of hepatitis b virus, antibodies to HIV, hepatitis C retroplatsentarno blood is centrifuged, add sodium chloride (0.1 to 1.0 mol/l) and Triton X-100 (0.01 and 0.1%) for blocking of nonspecific adsorption and inactivation of viruses, and again centrifuged. The thus prepared serum saturated with ammonium sulfate to 30%, incubated over night at +4°C and stirring and centrifuged. The precipitate washed three times 30% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride, and cialiswhat in the ultrafiltration cell, missing a 10-fold volume of buffer. The resulting diluted 5 times with 0.1 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05% Triton X-100, and subjected to ultrafiltration in the ultrafiltration cell with a filter to limit exceptions 100000 Yes. The resulting permit put on affinity sorbent with immobilized monoclonal antibodies to TBG. Nonspecific sorbiruyushchiesya components of the serum wash with sorbent consistently 4-5 volumes of 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride, and the same volume of 0.15 M sodium chloride solution. Affine-associated TBG elute with 0.1 M glycine-HCl buffer solution.

The eluate quickly neutralized to a pH of 6.5-7.5, dialist against saline and lyophilizers.

Crucial advantage of the proposed method is able to obtain the drug TBG with a high degree of purity and effective yield of the target product.

The method is illustrated by the following examples.

Example 1. All procedures performed at +4°C. 200 ml retroplatsentarno blood centrifuged with a speed of 10,000 rpm for 1 hour, the precipitate discarded, and the supernatant add sodium chloride to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.05%. The centrifugation repeated. Serum containing 31 mg TBG, thus prepared, is saturated with ammonium sulfate to 30% saturation, incubated overnight with stirring and cent is fugeret with a speed of 10,000 rpm for 1 hour. The precipitate washed three times 30% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05% Triton X-100, dialist in the ultrafiltration cell, missing a 10-fold volume of buffer. To the drug add 4 volumes of 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05% Triton X-100 and subjected to ultrafiltration in the ultrafiltration cell using a filter with a limit exceptions 100000 Yes to complete the passage of the solution through the filter. The resulting permeate is combined with 40 ml sepharose CL-6B, with "sewn" monoclonal antibodies to TBG. Incubation is carried out for 12 h with constant stirring on an orbital shaker.

Nonspecific sorbiruyushchiesya components of the serum wash with sorbent consistently 4-5 volumes of phosphate-saline buffer solution containing 0.5 M sodium chloride solution and the same amount of 0.15 M solution of sodium chloride. The sorbent is placed in a chromatographic column of 2.5×8,5 see After washing the sorbent solution of sodium chloride conduct elution with 0.1 M glycine-Hcl buffer solution with a pH of 2.55 at 80 ml/h, while in optical density at a wavelength of 280 nm. The elution is carried out to achieve zero values of optical density. The fraction containing the target product immediately neutralized to a pH of 6.5-7.5, concentrate, dialist in ultrafiltration cell relative to saline and lyophilizers.

The purity of the obtained product, analyzed by electrophoresis in a density gradient polyacrylamide gel, is 98%. The output of the preparation according to the immunoassay of 23.3 mg (75%). The drug test for the absence of antibodies to HIV, hepatitis b virus and antibodies to hepatitis C.

Example 2. All procedures performed at +4°C. 200 ml retroplatsentarno blood centrifuged with a speed of 10,000 rpm for 1 hour, the precipitate discarded, and the supernatant add sodium chloride to a final concentration of 0.5 M and Triton X-100 to a final concentration of 0.05%. The centrifugation repeated. Serum containing 31 mg TBG, thus prepared, is saturated with ammonium sulfate to 30% saturation, incubated overnight with stirring and centrifuged with a speed of 10,000 rpm for 1 hour. The precipitate washed three times 30% ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05% Triton X-100, dialist in the ultrafiltration cell, missing a 10-fold volume of buffer. To the drug add 4 volumes of 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05% Triton X-100, and all Laut ultrafiltration in the ultrafiltration cell using a filter with a limit exceptions 100000 Yes to complete the passage of the solution through the filter. The permeate is applied to the chromatographic column of 2.5×8,5 filled with 40 ml sepharose CL-6B, with "sewn" monoclonal antibodies to TBG, equilibriating phosphate-saline buffer solution. The rate of deposition of 40 ml/hour, a Drawing exercise three times the passage of the applied volume through the column.

Nonspecific sorbiruyushchiesya components of blood wash with sorbent consistently phosphate-saline buffer solution containing 0.5 M sodium chloride, and 0.15 M sodium chloride at a rate of 80 ml/h until reaching zero values of optical density.

After washing the sorbent is carried out the elution of 0.1 M glycine-HCl buffer solution with a pH of 2.55 and a rate of 40 ml/hour elution Process control by optical density at a wavelength of 280 nm. Elution is carried out until reaching zero values of optical density. The fraction containing the target product, immediately neutralized to a pH of 6.5-7.5, concentrate, dialist in ultrafiltration cell relative to saline and lyophilizers.

The purity of the obtained product, analyzed by electrophoresis in a density gradient polyacrylamide gel, is 98%. The output of the preparation according to the immunoassay is 25.1 mg (81%). The drug test for the absence of antibodies to HIV, hepatitis b virus and antibodies to the virus of hepatitis is itâ C.

Using the proposed method allows to obtain the target product with a high purity (at least 98%) and high: 75-81% yield. Step-by-step description of procedures for the isolation and purification are shown in table 1.

Table 1
Example 1Example 2
No.OperationVolume of sample (ml)Qty TBG (mg)Volume of sample (ml)Qty TBG (mg)
1Alpacamania fractionation10281028
2Ultrafiltration50275027
3Affinity chromatography8024,51002,9
4Concentration/dialysis/centrifugation5,623,35,825,1
Output75%81%

Sources of information

1. Aveskulov, Haasse, Nvenergy, Justiniano. Isolation and purification of specific β1-g-globulin. "Questions of medical chemistry, 1978, No. 24, s-244.

2. Swimers, Schrivener, Appalling, Usepagov, Justiniano. The method of obtaining the USSR beta-1-glycoprotein from waste retroplatsentarno blood in the production of gamma globulin. A.S. No. 1341736, priority 28.03.85.

3. Schrivener, Art, Lev, Ivekoviceva, PKa, Ackbarali, Justforyou way to obtain the USSR beta1-glycoprotein. A.S. No. 1783644, priority 04.12.89.

4. MeV, Schrivener, Justiniano. Comparative physico-chemical characteristics of drugs USSR β1-glycoprotein isolated from retroplatsentarno blood. J. "Bulletin of experimental biology and medicine, 1990, No. 9, s-267.

5. Wailing, Art, Justiniano. "A method of obtaining USSR sub> 1-glycoprotein". A.S. No. 1836957, priority 25.03.90.

The isolation and purification of USSR beta-1-glycoprotein (TBG) of serum retroplatsentarno blood by fractionation with ammonium sulfate, washing and centrifugation, dissolved sediment, affinity chromatography on sepharose with subsequent elution of the target product and its lyophilization, wherein retroplatsentarno blood centrifuged at 10,000 rpm for one hour, the supernatant add sodium chloride to a final concentration of 0.1-1.0 mol/l) and Triton X-100 to a final concentration of 0.01-0.1%, repeat centrifugation, the resulting serum is saturated with ammonium sulfate to 30%, incubated over night under stirring, centrifuged, the precipitate washed three times 30%ammonium sulfate, suspended in 0.15 M phosphate-saline buffer solution pH 7.0 and 7.5, containing 0.5 M sodium chloride and 0.05%Triton X-100, dialist in the ultrafiltration cell, missing a 10-fold volume of buffer solution, then diluted 5 times and spend ultrafiltration with a limit exceptions 100000 D, the resulting permeate is subjected to a single affinity chromatography on sepharose with immobilizovannymi monoclonal antibodies to TBG, elute ofinnovations TBG in 0.1m glycine-HCl buffer solution pH 2,55, the eluate is immediately neutralized, concentrated, dialist in the saline and lyophilizers.



 

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9 cl, 6 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to the impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. In case of no exudative phenomena on patient's eye bottom the obtained preparation should introduced at the quantity of 7-10 ml once in 48 h for 1 mo (totally, 15 injections). In case of exudative-hemorrhagic phenomena it should be introduced parabulbarly at the volume of 0.5 ml and parenterally - 7.0-10.0 ml once in 48 h for 1 mo per 15 injections, correspondingly. The preparation enables to improve visual functions due to decreased tissue hypoxia and normalization of microcirculation in visual analyzer.

EFFECT: higher efficiency of therapy.

2 cl, 7 dwg, 2 ex

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